An analysis chip comprises a substrate on which a selective binding substance (such as a nucleic acid, a protein, a lipid or a saccharide) that selectively binds to a test substance is immobilized. The selective binding substance on the substrate and the test substance are allowed to undergo a hybridization reaction usually in a solution and, from the results of the reaction, the existence, condition, quantity or the like of a substance contained in the test substance are analyzed. As the substrate, a glass substrate, a metal substrate or a resin substrate is usually employed.
One example of an analysis chip is called “microarray” in which molecules such as DNAs, proteins or sugar chains are densely arranged on a substrate for the purpose of, for example, simultaneously assaying the expressions of numerous genes in the number of several tens to several tens of thousands. The use of microarray enables detection and quantification of nucleic acids based on nucleic acid-nucleic acid hybridization reaction or detection and quantification of proteins and sugar chains based on protein-protein, sugar chain-sugar chain or sugar chain-protein specific reaction so that systematic and comprehensive gene expression analysis can be carried out on, for example, various disease animal models and cell biological phenomena. Specifically, the functions of genes, that is, proteins encoded by the genes can be clarified, and the timing of the expression of the proteins as well as the places of their actions can be identified. By using a microarray to analyze variations in gene expression of organisms at the cell or tissue level and combining the data of physiological, cell biological and biochemical phenomena to construct a database for gene expression profiles, it becomes possible to search disease genes and therapy-related genes and to explore therapeutic strategies.
Among analysis chips, DNA microarrays (DNA chips) are used for detection, quantification and the like of nucleic acids based on nucleic acid-nucleic acid hybridization reaction. As a DNA chip, for example, a chip in which a large number of DNA fragments are densely arrayed and immobilized on a glass flat substrate is employed. Such a DNA chip is used to detect each gene contained in a sample or measuring the amount thereof by, for example, a method in which a sample prepared by labeling the genes expressed in a cell of interest or the like with a fluorescent dye or the like is subjected to hybridization to allow complementary nucleic acids (DNA or RNA) to bind with each other and the fluorescence of the binding sites is quickly detected using a high-resolution detection device (scanner), or a method of detecting a response such as electric current based on an electrochemical reaction. DNA chips have large expectations not only in gene expression analysis based on detection and quantification of expressed genes, but also in its application fields such as detection of single nucleotide polymorphisms (SNP) in genes.
In addition, analysis chips have been utilized as a means of examining and analyzing not only nucleic acids such as DNA, but also proteins and saccharides. Especially, in protein analysis chips, proteins such as antibodies, antigens and enzyme substrates are immobilized on a substrate.
WO 2005/090997 discloses a method of stirring a test substance-containing solution by rotating an analysis chip having an irregular structure and thereby allowing fine particles or air bubbles to move in the analysis chip. In that method, by allowing the fine particles or air bubbles to move without coming into contact with the surface immobilized with a selective binding substance, even with a trace amount of the test substance, good S/N ratio and strong fluorescence signal can be obtained.
JP 2007-285828 A discloses a method capable of carrying out a selective reaction between a test substance and a selective binding substance in a simple and stable manner by rotating an analysis chip having an irregular structure in the substantially horizontal direction and stirring the test substance solution using fine particles.
JP 2003-339375 A discloses a hybridization method and an apparatus in which, by rotating a container containing a sample solution and fine particles and allowing the fine particles to fall in the direction of gravity, the sample solution in the container is stirred.
Japanese Patent No. 4473007 discloses a hybridization method wherein a hybridization solution is injected into a special hybridization chamber in which a microarray is arranged such that the space thereof is partially left unfilled and the chamber is then rotated to shift the position of the space filled with the solution in the chamber, thereby stirring the solution.
U.S. Pat. No. 6,309,875 discloses a rotation-and-revolution type hybridization apparatus which stirs a sample solution by rotating the apparatus itself while revolving a microarray arranged on a turntable.
In the method of stirring a solution according to WO 2005/090997, the analysis chip is rotated at a relatively low rotation rate of, for example, 3 rpm and, in that case, the hybridization reaction requires 10 hours. Therefore, that method is not suitable for prompt detection of a test substance. In the same manner, the method of stirring a test substance solution according to JP 2007-285828 A is also not applicable to prompt detection of a test substance because the hybridization reaction in that method requires 16 hours. Further, although the method disclosed in JP 2003-339375 A is stated to have an effect of shortening the time required for hybridization, the hybridization reaction actually requires 6 hours. Therefore, it is difficult to apply that method to an analysis where prompt diagnosis is demanded. Moreover, in the hybridization method disclosed in Japanese Patent No. 4473007, although the CV value is improved by rotating the chamber as compared to when the reaction is carried out by simply leaving the hybridization solution, there is hardly any change in the signal intensity and the progress of the reaction is not accelerated. The apparatus disclosed in U.S. Pat. No. 6,309,875 is an apparatus which enables stirring of microarray with a small amount of sample solution. However, the time required for hybridization and shortening thereof are not mentioned and it is thus unclear if the apparatus is adaptable to prompt diagnosis.
The solution-stirring methods disclosed in WO 2005/090997, JP 2007-285828 A, JP 2003-339375 A, Japanese Patent No. 4473007 and U.S. Pat. No. 6,309,875 are all aimed at improving the detection sensitivity by increasing the efficiency of hybridization reaction. However, hybridization reactions in those methods actually require 6 to 20 hours. Thus, those methods cannot be viewed as technologies to dramatically improve the speed of detection or quantification of a test substance using an analysis chip. Therefore, until now, in the field of analysis of a test substance using an analysis chip where it is demanded to perform detection or quantification in a short time of several minutes to two hours at the most, for example, in the examination and diagnostic applications of infectious diseases such as influenza, sepsis and the like, there has not been presented a method of stirring a test substance solution which enables analysis to be performed with such a speed that satisfies the demand.
It could therefore be helpful to provide a means of accelerating the progress of selective binding reaction (hybridization reaction) between a selective binding substance immobilized on an analysis chip and a test substance, particularly a means of enabling analyzation of a test substance in a short time.